Translation of the genetic information in the cell into functional proteins is an essential and highly conserved function. The ribosome, a two-subunit macromolecular complex, composed in bacteria of three large RNAs (rRNAs) and more than 50 proteins (r-proteins), is the catalyst and framework for the intricate and coordinated process of translation. Peptidyl transferase, likely the most primitive activity of the ribosome, is of central importance. Theoretical considerations of the origin of life predict a central role for the rRNAs in translation, a prediction strengthened by the demonstration of catalysis by RNA and consistent with the extreme phylogenetic conservation observed among rRNA sequences. Extensive biochemical and genetic studies indicate that the rRNAs play a primary functional role in the processes of translation, and in particular 23S rRNA in the central catalytic event, peptide bond formation. Structural studies of the ribosome, including cryoelectron microscopy and X-ray crystallography, are yielding a wealth of information. However, the static views of the ribosome yielded by such approaches will not reveal the mechanism or dynamics of the intricate process of translation. This proposal describes in vitro genetic and biochemical approaches to identify the molecular components at the active site of peptidyl transferase (RNA or protein). The goal of these studies is to understand how these components conspire in the formation of peptide bonds and to understand how this activity is controlled between each catalytic cycle to allow for the generation of high-fidelity encoded protein products. First, site-directed mutagenesis and modification interference approaches will be used to identify nucleotides in 23S rRNA that are specifically involved in tRNA substrate binding and catalysis by the ribosome. Second, a recently developed iterative in vitro selection approach will be used to optimize the activity of ribosomes reconstituted from in vitro transcribed Bacillus stearothermophilus 23S rRNA. Finally, a variety of biochemical approaches including crosslinking, kinetic studies and chemical modification will be used to characterize the structure and function of the hybrid state of tRNA binding in translational elongation. Information obtained from these studies will eventually help to define the features of current ribosome- specific antibiotics which make them effective and the mechanism by which these same antibiotics are evaded by the host. Ultimately, this will lead to the rational design of novel antibiotics.